This study aimed to investigate how 6-bromoindirubin-3'-oxime (BIO) increases the osteogenic differentiation of canine bone mesenchymal stem cells (BMSCs) and the role of the Wnt/β-catenin signaling pathway in this process. We mimicked the effect of Wnt by adding BIO to the culture medium of BMSCs and examined whether canonical Wnt signaling positively affects the differentiation of these cells into osteoblasts. Canine BMSCs were cultured with 0.5 and 1.0 μM BIO under osteogenic conditions and then differentiation markers were investigated. It was found that BIO significantly increased the activity of alkaline phosphatase (ALP), the number of ALP-positive cells, the mineralization level and calcium deposits. Moreover, cells cultured with 0.5 and 1.0 μM BIO exhibited detectable β-catenin expression in their nuclei, and showed upregulated β-catenin and glycogen synthase kinase 3 beta(GSK3β) phosphorylation compared to untreated cells. In addition, BIO enhanced the mRNA expression of osteoblast differentiation markers such as ALP, runt-related transcription factor 2, collagen I, osteocalcin, and osteonectin. In conclusion, BIO upregulated GSK3β phosphorylation and inhibited its activity, thereby activating the Wnt/β-catenin signaling pathway and promoting the osteogenic differentiation of canine BMSCs. The effect of 1.0 μM BIO on BMSCs differentiation was stronger than that of 0.5 μM BIO.